O. J. H. M. Verhagen

Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes

Analysis of minimal residual disease (MRD) can predict outcome in acute lymphoblastic leukemia (ALL). A large prospective study in childhood ALL has shown that MRD analysis using immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets can identify good and poor prognosis groups of substantial size that might profit from treatment adaptation. This MRD-based risk group assignment was based on the kinetics of tumor reduction. Consequently, the level of MRD has to be defined precisely in follow-up samples. However, current PCR methods do not allow easy and accurate quantification. We have tested ‘real-time’ quantitative PCR (RQ-PCR) using the TaqMan technology and compared its sensitivity with two conventional MRD-PCR methods, ie dot-blot and liquid hybridization of PCR amplified Ig/TCR gene rearrangements using clone-specific radioactive probes. In RQ-PCR the generated specific PCR product is measured at each cycle (‘real-time’) by cleavage of a fluorogenic intrinsic TaqMan probe. The junctional regions of rearranged Ig/TCR genes define the specificity and sensitivity of PCR-based MRD detection in ALL and are generally used to design a patient-specific probe. In the TaqMan technology we have chosen for the same approach with the design of patient-specific TaqMan probes at the position of the junctional regions. We developed primers/probe combinations for RQ-PCR analysis of a total of three IGH, two TCRD, two TCRG and three IGK gene rearrangements in four randomly chosen precursor-B-ALL. In one patient, 12 bone marrow follow-up samples were analyzed for the presence of MRD using an IGK PCR target. The sensitivity of the RQ-PCR technique appeared to be comparable to the dot-blot method, but less sensitive than liquid hybridization. Although it still is a relatively expensive method, RQ-PCR allows sensitive, reproducible and quantitative MRD detection with a high throughput of samples providing possibilities for semi-automation. We consider this novel technique as an important step forward towards routinely performed diagnostic MRD studies.

Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia

Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that ‘real-time’ quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline JH TaqMan probes to be used in combination with one of six corresponding germline JH primers and one allele specific oligonucleotide (ASO) primer complementary to the junctional region. In nine cases in which both approaches were compared, at least similar (n = 4) or slightly higher (n = 5) maximal sensitivities were obtained using an ASO primer. The ASO primer approach reached maximal sensitivities of at least 10−4 in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions had to be increased to remove or diminish background signals; this only concerned the frequently occurring JH4, JH5 and JH6 gene rearrangements. After optimization of the conditions (mainly by increasing the annealing temperature), only occasional aspecific amplification signals were observed at high threshold cycle (CT) values above 42 cycles and at least six cycles above the CT value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline JH TaqMan probes and six corresponding germline JH primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies.

Noninvasive fetal genotyping of human platelet antigen-1a

Please cite this paper as: Scheffer P, Ait Soussan A, Verhagen O, Page‐Christiaens G, Oepkes D, de Haas M, van der Schoot C. Noninvasive fetal genotyping of human platelet antigen‐1a. BJOG 2011;118:1392–1395.

Suitable DNA isolation method for the detection of minimal residual disease by PCR techniques.

Suitable DNA isolation method for the detection of minimal residual disease by PCR techniques

Rearrangement status of the malignant cell determines type of secondary IgH rearrangement (V-replacement or V to Dj joining) in childhood B precursor acute lymphoblastic leukemia

Immunoglobulin heavy chain (IgH) oligoclonality in childhood B precursor acute lymphoblastic leukemia (ALL) as determined by Southern analysis is found in 30–50% of patients and has been shown to be the result of ongoing IgH rearrangement (mostly VH-replacement and VH to D–JH joining) after malignant transformation. It is unknown however, what determines the type of secondary rearrangement. Also the biological basis of the variable degree of oligoclonality observed in childhood ALL is poorly understood. We analyzed in detail the IgH rearrangement status of the leukemic cells for a random panel of 18 childhood B precursor ALL patients by polymerase chain reaction (PCR)/sequencing analysis and by Southern analysis. By Southern analysis 10/18 (55.6%) patients were considered oligoclonal and 8/18 (44.4%) monoclonal. In contrast, by PCR minor clonal rearrangements were detected in 14/18 (77.8%) patients. VH-replacement was found in 7/14 patients, VH to D–JH joining in 6/14 patients and an unusual type of secondary rearrangement, VH–D to JH joining, in one patient. Only a single type of secondary rearrangement was detected in each patient. The type of secondary rearrangement (VH-replacement or VH to D–JH joining) depended on the rearrangement status (VDJ/VDJ or VDJ/DJ, respectively) of the dominant leukemic clone as determined by Southern analysis. We found that in addition to a more ‘advanced’ IgH rearrangement status patients with VH-replacements also have a more ‘advanced’ TCRδ rearrangement status, which possibly reflects exposure of both the IgH locus and the TCRδ locus to recombinase activity in a preleukemic clone. Finally, we investigated a putative relationship between oligoclonality by Southern analysis and S-phase fraction of the leukemic cell population. We found a significantly lower percentage cells in S-phase for oligoclonal patients as compared to monoclonal patients. Our data add to the understanding of ongoing rearrangement of antigen receptor loci in childhood ALL and have implications for the monitoring of minimal residual disease by PCR.

The analysis and quantification of a clonal B cell response in a hyperimmunized anti-D donor

Healthy volunteers are hyperimmunized with RhD‐positive red cells in order to obtain plasma containing high titres of anti‐D immunoglobulin, which is used for the prevention of haemolytic disease of the fetus and newborn. We analysed the anti‐D immune response in a donor who had been hyperimmunized for 7 years and who showed declining anti‐D titres despite re‐immunization. A phage display library representing the complete immunorepertoire and a second library representing the IGHV3 superspecies family genes (IGHV3s) repertoire in the donor were constructed and analysed. A clonal Ig‐gene rearrangement was quantified in the peripheral blood by limiting dilution polymerase chain reaction (PCR) All RhD‐binding phages from both libraries, except one, had heavy chains with IGH–VDJ rearrangements of the same clonal origin, but with different patterns of somatic mutations and joined with different light chains. Limiting dilution PCR performed on mRNA and genomic DNA showed a frequency of 1 clonal B cell in 2000 IgG1/3‐positive B cells. We show the presence of clonally related RhD‐specific B cells in a hyperimmunized anti‐D donor who had declining anti‐D titres and who was unresponsive to re‐immunization. Furthermore, we found a high frequency of clonal B cells. These results contribute to the understanding of the immune response against RhD in hyperimmunized anti‐D donors.

Detection and Quantification of Minimal Residual Disease in Childhood B-Precursor Acute Lymphoblastic Leukemia by Limiting Dilution and PCR Application

Polymerase Chain Reaction (PCR) on Immunoglobine heavy chain and T-cellreceptor can be applied for the detection of Minimal Residual Disease (MRD) in children with B-precursor Acute Lymphoblastic Leukemia (ALL). We have previously shown that the presence of PCR detectable malignant cells at the end of induction therapy is strongly correlated with occurrence of a relapse. We currently investigate whether the predictive value of PCR can be increased by quantification of MRD during induction therapy.